1. Field of the Invention
The present invention relates to a method for producing a polyhydroxyalkanoic acid (PHA), and more particularly, to a method for producing a polyhydroxyalkanoic acid containing a high content of long-chain aromatic monomer-units by growing a phaZ mutant of a Pseudomonas strain in a medium containing a sugar, a substituted fatty acid, and a salicylic acid.
2. Description of the Related Art
Polyhydroxyalkanoic acids (PHAs) are polyesters accumulated as an intracellular carbon and energy storage material in various microorganisms under unbalanced growth condition of limiting nutritional elements such as phosphorous, nitrogen, magnesium, and oxygen in the presence of excess carbon source. PHAs have attracted much attention as promising substitutes for chemically synthesized polymers due to their similar mechanical properties to petroleum-derived plastics and complete biodegradability.
PHAs are accumulated in a wide range of microorganisms in the form of granular inclusion bodies. The PHAs that microorganisms produce are divided into two classes, short-chain-length (SCL-) PHAs (C4 and C5) and medium-chain-length (MCL-) PHAs (C6-C14). MCL-PHAs have attracted attention because of the possible incorporation of many functional groups, such as phenyls, phenoxy groups, alkenes, etc., into their side chains, thereby resulting in the improved properties such as the increased transparency. For example, the MCL-PHA containing a monomer derived from MCL fatty acids having an aromatic substituent at the co-position may be used as a starting intermediate in pharmacology and in various fields.
MCL-PHA producing Pseudomonas spp. are typical bacteria that can produce aromatic polyesters from phenyl group carboxylic acids. However, some precursors containing a phenyl group or modified phenyl group, such as 5-phenylvalerate, (5PV), 5-(4-tolyl)-valerate, etc., are not readily utilizable by bacteria when they are fed as the sole carbon source. In order to increase the utilization capability of recalcitrant carbon compounds, the cometabolism method has been suggested. Most studies have is employed the cometabolism method in which a good polymer-producing substrate (such as either octanoic acid or nonanoic acid) has usually been used as a cosubstrate. Accordingly, since the resulting polymer obtained by the cometabolism method is a mixture of an aromatic homopolymer, an aromatic/aliphatic copolymer, and an aliphatic polymer, the isolation and purification of pure aromatic homopolyester from the recovered mixed polymer sample requires a tedious fractionation procedure using repetitive solution precipitation steps.
Therefore, it has been necessary to develop a technology for producing pure aromatic polyester in quantity. It has been seen that some carboxylic acids having low carbon number (C2-C5), fructose, and glucose in Pseudomonas putida BM01 do not induce PHA production but support the growth of a large amount of cells. The use of carbon sources in cometabolism with w-phenylalkanoic acids or 11-phenoyundecanoic acids (11-POU) resulted in a high production of pure aromatic polyesters free from aliphatic monomer-units in Pseudomonas putida BM01.
Inhibitors for PHA synthesis may be used to carry intermediates in a specific metabolic pathway to the PHA synthesis pathway. Acrylic acid is known as a β-oxidation inhibitor for bacteria, and when Ralstonia eutropha, which is a typical bacterium that accumulates SCL-PHA, was grown using octanoic acid as a carbon source, the acrylic acid was successfully used to introduce the MCL monomer as a comonomer to the PHA. It is known that 2-bromooctanoic acid (2-BrOA) does not affect cell growth on sugars but inhibits only MCL-PHA accumulation in Pseudomonas spp. Therefore, 2-BrOA effectively inhibits the production of PHA composed of (R)-3-hydroxyacyl monomers, induced by ((R)-3-hydroxyacyl)-ACP 4 CoA transferase (PhaG) from the coadded, unrelated carbon source such as sucrose, it can be effectively used in the preparation of PHA which is functionally designed from carboxylic acid having a functional group in Pseudomonas spp.
Recently, another inhibitor for the production of MCL-PHA has been reported. When Pseudomonas aeruginosa BM114 capable of accumulating both SCL-PHA and MCL-PHA was grown in a medium-chain carboxylic acid (e.g., C8, C9 and C10 carboxylic acids), while the treatment of acrylic acid just inhibited the production of SCL-PHA, the treatment of salicylic acid just inhibited the production of MCL-PHA.
The material properties of MCL-PHA depend on the length and uniformity of spacer side-chain with a functional pendent group. Thus, modulation of the distribution of side-chain monomer-units is expected to improve material properties.
Generally, the monomer distribution in MCL-PHA depends on the specificity of PHA synthase and the intracellular concentration of monomer precursors. When MCL fatty acids are used as carbon source, the monomer precursors are mostly supplied through enzyme(s) (e.g., enoyl-CoA hydratase (PhaJ) linking the β-oxidative pathway and the PHA synthesis pathway. In the previous study of the inventors of the present invention, under a synthetic strategy that combined the β-oxidation inhibition by salicylic acid and cometabolism of 11-POU and fructose, Pseudomonas fluorescens BM07 was able to shift the distribution of aromatic medium-chain-length monomer-units in the PHA to longer units.
Thus, salicylic acid could be an efficient mediator in producing longer monomer precursors like 3-hydroxy-9-phenoxynonanoate (9POHN), twice as much as in a simple cosubstrate method. Since the cell growth is insignificant in a medium containing only 11-POU as a single carbon source in the presence of salicylic acid, the significant increase by salicylic acid was possible only by the cosubstrate metabolism using fructose and 11-POU.
Intracellular PHA depolymerase PhaZ is responsible for the degradation or intercellular PHA granules to supply the hydrolyzed monomer energy and other ingredients in cells. Deletion of phaZ in Pseudomonas putida KT2442 has been known to increase the accumulation of PHA in the cell when the mutant cell was grown on octanoate. Meanwhile, disruption of phaZ gene was expected to affect intracellular levels and half-lives of β-oxidation intermediates, eventually affecting the rate of incorporation of 3-hydroxy-monomer-units into PHA from the coadded functional fatty acids (e.g., 11-POU). In the present invention, the phaZ gene in P. fluorescens BM07 was disrupted using an insertional mutagenesis technique and the effect of salicylic acid on the shifting of aromatic monomer-unit distribution as well as the production of the aromatic PHA in the mutant was investigated.
The inventors of the present invention have found that the salicylic acid (1 mM) treatment significantly increased the level of longer aromatic monomer-units and the yield of conversion of 11-POU to PHA in BM07-ΔphaZ mutant. In addition, the phaZ gene was over-expressed in the wild-type strain to assess how PhaZ affects the comonomer composition of aromatic PHA. Enhancement of the conversion yield by salicylic acid was also observed for other types of aromatic carboxylic acids such as 5-phenylvalerate (5PV) and 6-phenylcaproate (6PC).